Botulinum neurotoxin (BoNT) is a protease naturally produced in a pathogenic bacterial strain, Clostridium botulinum. Food borne botulism is primarily due to contamination of food by bacterial spores in an anaerobic environment. The bacteria require an anaerobic environment for the spores to germinate and for bacteria to grow. Botulism can be life threatening. The protein acts as a neurotoxin and is the most acutely toxic substance known to man. However, the toxin has legitimate medical uses for treating diseases including diseases associated with severe muscle spasms, hyperhidrosis, pain, and others as the toxin can inhibit nerve activity by blocking neurotransmitter transmission, thereby decreasing muscle activity. The most lucrative application of BoNT is cosmetic usage where companies again harness the toxin's ability to decrease muscle activity thereby smoothing wrinkles.
Botulinum neurotoxin is classified by the Centers for Disease Control and Prevention as one of the six highest-risk threat agents for bioterrorism (“Category A” bioterrorism agents). BoNT is a 150 kDa single chain protein which is linked by a disulfide bond that can be cleaved into a 100 kDa Heavy Chain (HC) and a 50 kDa Light chain (LC) by endogenous or exogenous proteases. The LC is a zinc metalloprotease which cleaves different SNARE proteins depending on the serotype causing flaccid paralysis. The HC consists of a C-terminal binding domain (HC) which is responsible for receptor binding and a N-terminal translocation domain (HN) which is responsible for delivering the catalytic light chain to the neuronal cytosol. Based on their ability to be neutralized by type specific antiserum, BoNTs have traditionally been categorized into seven serotypes (BoNT/A-G), among which BoNTs A, B, E, and F are known to cause human botulism.
An in vivo mouse bioassay is an FDA-approved method for detecting BoNT activity. See Hatheway et al., in Laboratory Diagnosis of Infectious Diseases: Principles and Practice. Springer-Verlag, New York, 1988, p. 111-133; Schantz and Kautter, J. Assoc. Off. Anal. Chem. 61:96-99 (1978). The mouse bioassay, which detects low pM amounts of BoNT/A, requires injecting mice intraperitoneally or intravenously with toxin or toxin/antibody mixtures and observing the injected mice for signs of toxicity and death. Another FDA-approved method for detecting BoNT activity is a cell-based assay using SiMa cells. See Fernández-Salas et al., PLoS One 7(11):e49516 (2012). For in vitro assays, continuous cell lines such as Neuro-2a, SK-N-SH, M17, SH-SY5Y, NT2, PC12, and SiMa have been tested for sensitivity to BoNT/A and are being used as research models. These cell lines, however, generally are insufficiently sensitive to compete with the mouse bioassay with the exception of differentiated SiMA cells (Fernández-Salas et al., supra). Accordingly, there remains a need for practical and highly sensitive compositions and methods for detecting botulinum neurotoxin and their neutralizing antibodies for clinical and research applications.